The Tat protein of the human immunodeficiency virus (HIV) is a protein produced early in infection and is required for efficient replication of the virus. Tat shows very little variation between HIV subtypes. Tat is released from cells at relatively high levels and can be detected in the serum of HIV infected individuals. Intracellular Tat is efficiently processed by major histocompatibility complex (MHC) class I for presentation to cytotoxic T lymphocytes (CTL). Several groups have advanced the idea that Tat would make a logical prophylactic vaccine candidate and that both neutralizing antibody and CTL responses are needed. The overall goal of our research is to develop nanoparticle-based HIV-1 Tat vaccines to elicit enhanced Th1, CTL, and humoral immune responses with nanoparticles over either adjuvanted protein or 'naked' plasmid DNA alone. The specific goal of this 24 month Research Plan is to demonstrate that two different types of nanoparticle-based HIV-1 vaccines can be engineered from novel microemulsion precursors to deliver either recombinant Tat (rTat) or plasmid DNA expressing Tat (pDNA-Tat) to dendritic cells (DCs) after either topical application or subcutaneous injection in mice. Stable anionic nanoparticles (type 1) or cationic nanoparticles (type 2) will be engineered from novel microemulsion precursors using an inexpensive, reproducible, and scalable process and used for coating by rTat and pDNA-Tat, respectively. The two Specific Aims are as follows: Specific Aim #1: Preparation of Nanoengineered HIV-I Vaccines. i) demonstrate that anionic nanoparticles can be coated with rTat, and that cationic nanoparticles can be coated with pDNA-Tat, ii) demonstrate that a dendritic cell-specific ligand, mannan, can be coated on both types of nanoparticles, and iii) characterize the nanoparticle-based vaccines in terms of particle size, surface charge, stability, and cell uptake and/or transfection in human dendritic cells. Specific Aim #2: Testing of Nanoengineered HIV-1 Vaccines in Mice. i) demonstrate nanoparticle targeting to DCs in-vivo and test adjuvanticity of prototype nanoparticles in-vitro, ii) demonstrate enhanced Th1, CTL, and humoral immune responses with these mannan-coated nanoparticle vaccines over either ALUM-adjuvanted rTat or 'naked' plasmid DNA after both topical and subcutaneous administration, and iii) demonstrate that a heterologous prime-boost regimen of nanoparticle-based HIV-1 vaccines using rTat and pDNA-Tat is superior to a homologous regimen using either rTat or pDNA-Tat immunization alone. This proposal relates to novel and innovative vaccine discovery and development concepts of dendritic cell-targeted nanoparticles containing either rTat or pDNA-Tat, and the use of Tat (1-72) to generate protective Th1, CTL, and humoral responses. The development of an appropriate nanoparticle-based HIV-1 Tat vaccine will also benefit strategies that may incorporate multiple HIV genes such as Tat, Env, Gag, Pol, and Rev.